Further investigations of the 5-HT in cultured cerebrovascular endothelium and isolated cerebral microvessels were concerned with its presence, derivation and/or control. 1) The level of 5-HT in the endothelial cultures could be decreased by incubation with either p-chlorphenylalanine (PCPA), the inhibition of tryptophan hydroxylase or by the addition of indomethacin, the inhibitor of prostaglandin synthesis. Moreover, the indomethacin-induced reduction of endothelial 5-HT was recoverable with the addition of prostacyclin. 2A) The isolated cerebral microvessels obtained either from saline-perfused or unperfused brains of rats pretreated with pargyline [monoamine oxidase (MAO) inhibitor] contained 8.6 and 18.9 pmoes/mg protein, respectively. 2B) The immunofluorescence studies of isolated cerebral microvessels revealed a diffuse and chainlike pattern of immunofluorescence specific for 5-HT. These findings clearly indicate that the 5-HT present in the microvessels is not only derived from serotonergic nerve endings but it is also formed in the endothelium where its level is most probably controlled by prostaglandin.